JPS61137887A - 2'-deoxyaristeromycin derivative and its use - Google Patents

Abstract

NEW MATERIAL:A compound of formula I [R<1> is H, amino group-protecting group; R<2> is H, spacer residue, hydroyl-protecting group, (protected) phosphoryl, (protected) deoxynucleotide chain bonding in the 5'-position; R<3> is H, hydroxyl- protecting group, (protected) deoxynucleotide bonding in the 3'-position; R<1>, R<2>, R<3> are not hydrogens at a time]. EXAMPLE:N<6>-Benzoyl-2'-deoxyaristeromycin. USE:A starting material for syntherizing oligodeoxynucleotide containing 2'- deoxyaristeromycin. It cause of depurination from the nucleotide under such acidic conditions as deprotection. It can be applied advantageously to the solid- phase synthesis. A reagent for genetic engineering. PREPARATION:For example, in accordance with reaction equations, aristeromycin (1) is benzoylated, the resultant tetrabenzoyl derivative (2) (A is formula II; Bz is benzoyl) is partially hydrolyzed and the hydroxyls in the 3' and 6'-positions are selectivity protected by silylation, then the hydroxyl in the 2'-position is converted into thioimdazolyl group, then the product is reduced and desilylated to give the exemplified compound.

Description

[Detailed description of the invention]

The present invention relates to 2'-deoxyaristeromycin derivatives and their uses. Furthermore, the present invention provides an advantageous method for chemically synthesizing oligodeoxynucleotides (2'-deoxyaristeromycin derivatives, which are raw materials for manufacturing 2ml), as well as incorporation into genes in the field of genetic engineering technology. Conventional technology In line with recent advances in gene manipulation technology, oligodeoxynucleotides (hereinafter referred to as
Chemical synthesis of oligomers (sometimes simply referred to as oligomers) has become popular (-). As a result, peptides and proteins that are useful to humans but could only be obtained in trace amounts from natural materials have been produced. With the help of microorganisms, it has reached a stage where it can be produced in factories.Such chemically synthesized oligomers have become indispensable for genetic manipulation, but their chemical synthesis is difficult to achieve with the mainstream solid-phase synthesis method. ,
It (2) In addition, liquid phase synthesis is also carried out. General (
-1 The solid-phase method is said to have many advantages over the liquid-phase method, such as simpler synthesis operations, better automation, and the need for a smaller amount of reagents for the condensation reaction. ing. The solid phase synthesis method uses, for example, a polystyrene resin or silica gel as a carrier, and uses a starting material 7 which has an ester bond with the 3' position of the 2'-deskin nucleon through a spacer such as succinic acid. A hydroxyl protecting group at the 5' position, such as dimethoxytrityl (DMT), is removed under acidic conditions to lead to a deprotected intermediate, and the desired base sequence of f'J sesame is added to the hydroxyl group at the 5' position. Accordingly, 1 to 8 protected nucleotide blocks are sequentially condensed. The problem with the above solid-phase synthesis method (2) is that while repeating the step of deprotecting under acidic conditions to obtain a 5'-human mixyl compound (2), the so-called depurination reaction in which the base part of the nucleotide is eliminated occurs. This is especially noticeable at the 3' end due to the presence of deoxyadenone (J. Stawinski et al. - Nucl, Ac1ds Red).
I, 4*353 (1977)). Furthermore, it has recently been pointed out that depurination occurs under acidic conditions when deoxyadenone is located at the 3'-terminus (not only at the 2-position, but also in a small number of cases where it is located in the oligomer chain). (T, Tanaka, R,L)
, Letsinger. Nucle+c Ac1d Re5earch, 1
0, 3249 (1982)). These by-products are difficult to remove during isolation and purification of the desired oligomer, and they not only shorten the chain length of the oligomer during the purification process, reducing the purity of the desired product, but also cause vector When carrying out genetic manipulation experiments7, such as those carried out by researchers, there is a high possibility that genes may be mistakenly incorporated and result in genes with base sequences different from the intended one. In order to prevent the production of such by-products (M, Sm
1th et al., J. Am. Chem, 5ac0.84, 4: J O (1962
), K. Jayaraman et al., Tetrahedron L
ett,, 23. 5377 (1982), T. P. Patel et al., Nu
cl. Ac1ds Res, 10, 5605 (1982
), M. J, Ga1tr:), Chem, Commun,
, 1982, 37°T+Tanaka et al., Nuc
l+Aclds Res,, 10. 3249 (1982) and Lewis acids such as zinc bromide! Efforts have been made to use (M, H. (:: aruthers et al., U, S, Pat, 126.
025 Feb. 29 1980 (C0A, 9 mutuals, P7015f). Kohli et al., Tetrahedron Lett,
, 21, 2683゜(1980), F, Ch.
ow et al., Nucl, Ac1dsRes, 9,2
807 (1981), etc.). However, it subsequently became clear that when a Lewis acid is used, a considerable amount of the protecting group at the 5' position remains due to incomplete reaction (M, S, Urd
ea et al., Proc. Nap. Acad, 8ci, USA, 80, 7461
(1981J), has become a serious synthetic obstacle. As explained above, 2′-
Chemical Synthesis of Oligodeoxynucleotides Containing Demoxyadenonine C2 1. There is no known definitive method to prevent depurination. For this reason, in the field of genetic engineering technology, the need for oligodeoxynucleotides as described above poses a major obstacle to the efficient progress of research and development. Means for Solving the Problems In view of the above problems, the present inventors have developed alisteromycin (T, Kusaka et al.,
J. Antibiotics, 21, 255
(1968) J is the isostere of adenosine (1so
stere), and after deriving it into a 2'-deoxy form, we synthesized an oligomer in place of 2'-deoxyadenosine, and found that the depurination reaction did not occur even under strongly acidic conditions. In addition, it was discovered that the obtained oligomer is similar to natural oligomer (= it can be used for genetic manipulation), and the present invention was completed through further research. (In the formula, R1 is hydrogen or an amino group protecting group '!', R''
is hydrogen, a hydroxyl group-protecting group, a spacer residue, an optionally protected phosphoryl group, or an optionally protected deoxynucleotide chain bonded at the 5' position, R8 is hydrogen,
Indicates a hydroxyl-protecting group or an optionally protected deoxynucleotide chain bonded at the 3' position, and R1, R2 and R3 are simultaneously (excluding when dihydrogen) 2
'-deoxyaristeromycin derivative, (wherein R1 is hydrogen or an amino group-protecting group, R2 is hydrogen or an optionally protected deoxynucleotide chain bonded at the 5' position, R is hydrogen or a hydroxyl group-protecting group) A 2'-deoxyarysterominone derivative represented by [indicating an optionally protected deoxynucleotide chain linked at the 3'-terminus] is linked at the 3'-terminus via a spacer. and 3) an oligodeoxynucleotide in which part or all of 2'-deoxyadenosine has been replaced with 2'-deoxyaristeromycin. The hydroxyl-protecting group in R2, the spacer residue, and the hydroxyl-protecting group in R3 include those normally used in the chemical synthesis of oligodeoxynucleotides, examples of which are as follows: As the amino group-protecting group, carbon Examples include aliphatic acyl groups having 1 to 20 carbon atoms and aromatic acyl groups having 7 to 10 carbon atoms. Specific examples include acetyl, imbutyryl, trinotylacetyl, and halogenoacetyl (e.g., trifluoroacetyl, gloloacetyl, etc.) , alkoxyacetyl (e.g., trityloxyacetyl, phenoxyacetyl, methoxyacetylnato), alkoxycarbonyl (e.g., valanitrophenoxycarbonyl, imbutyloxycarbonyl, tribromoethoxycarbonyl, paranitrophenylethoxycarbonylnato), hendiyl , anisoyl, etc.Next, as the hydroxyl group-protecting group, the same as the above-mentioned amino group-protecting group (in addition, alkylsilyl having 3 to 10 carbon atoms (e.g.,

[-butyldimethyldulyl, etc.], carbon number 5~
8 tetrahydropyranyl (e.g., tetrahydropyranyl, metquine tetrahydropyranyl, etc.), carbon number 3-1
O's alkoxyalkyl (e.g., ethoxyethyl, methoxyethyl, etc.), trityl and its substituted products (e.g., monomethoxytrityl, dimethoxytrityl, etc.) are exemplified.
Dibasic acids represented by H (n = 2 to 5), such as succinic acid, geltaric acid, and adipic acid, are exemplified, with succinic acid being the most preferred.Furthermore, a phenylthioethyl phosphate ester bond is also used as a spacer. It is possible, and the bond C-dipolyethylenediamine or ethylenediamine-glycine condensate may be interposed by an amide bond to form a part of the spacer. As a protecting group for the phosphoryl group, ortho or para-
Groups commonly used in polynucleotide synthesis (-) such as chlorphenyl, cyanoethyl 2.2.2-trichloroethyl, ethylthio, substituted or unsubstituted anilino, etc. A good deoxynucleotide chain is one consisting of one or up to about 150 deoxynucleotides containing any of the nucleobases adenine, guanine, cytosine, and thymine; If it contains, part or all of it may be replaced with 2'-deoxyaristeromycin. Here,
An optionally protected deoxynucleotide chain refers to a deoxynucleotide chain in which the amino group of the base portion is protected with a benzoyl or imbutyryl group, and the phosphoric acid between the nucleotides is protected with a group similar to the above-mentioned protecting group for the phosphoryl group. Show good. Next, the compound of general formula [1] is alisteromycin or 2'-deoxyaristeromycin (Japanese Patent Application Laid-open No.
62992λ as all starting materials, it can be produced by the following method (for example, 2'-deoxyaristeromycin is treated with the above protecting group as an acid chloride or anhydride, and 3
The hydroxyl groups at the ′ and 6′ positions and the amino group at the 6th position are simultaneously removed (unionized) and treated with a strong alkali such as alkali metal hydroxide (e.g., sodium hydroxide, potassium hydroxide, etc.) to selectively remove the hydroxyl protecting group. Next, the hydroxyl group at the 6' position is protected.In the case of a protecting group other than tetrahydropyranyl, this protection method is carried out in the presence of a basic catalyst such as dimethylaminopyridine or triethylamine in pyridine or an organic solvent. This is carried out by allowing a protecting group to act as a chloride.On the other hand, in the case of a tetrahydropyranyl group, it is introduced by addition in the presence of a dihydropyranyl acid catalyst, etc. Next, a hydroxyl group at the 3' position is added. A spacer is introduced into the DNA oligomer, which is achieved by, for example, using a dibasic acid as an anhydride in pyridine.On the other hand, an optionally protected phosphoryl group can be introduced into a synthetic raw material for DNA oligomers. As generally used (two methods used in the preparation of complete protection) (e.g., S, A, Navang, H, M, Hs+ung, RlBrousseau)
: MethodsEnzymo! ,, 68, 90
(1979) ) is applied. Further (-method called phosphorous acid ester method (for example, L, J 1McBr
ide 1M, HoCaruthers. Tetrahedron Lett, 245 (1
982) ) 'ze can also be used as a trivalent phosphorus compound intermediate. Specifically (the second is para-chlorophenyl phosphoric acid dichloride! The corresponding azolide (aZOI
After converting into ideJ, it is synthesized by reacting with a compound of the general formula (IJ in which R2 is hydrogen) and further reacting with β-cyanoethanol in the presence of a catalyst such as methylimidazole. Below, general formula (T) Regarding the method for producing the compound of R1
is a benzoyl group (Bz), R'' is a succinic acid residue, R3
A specific example in which is a dimethyltrityl group (DMT) will be described below with reference to a reaction process chart. Aristeromycin (1)'a = minipyridine benzoylated to obtain a tetrabenzoyl form (27). (2) was subjected to partial hydrolysis to form H6-benzoylaristeromycin (3), and then the 3' and 6' positions The hydroxyl group at the 2'-position of the compound (4) is selectively protected by nlylation to obtain cyclic nlyl ether (4).The hydroxyl group at the 2' position of the compound (4) is then thiocarbonylimidazolylated (5) and then α. d-azobisiso Reduction using tributyltin hydride in the presence of butyronitrile (AIBN) yields the 2'-deoxy compound (6).In addition to thiocarbonyldiimidazole, phenoxythiocarbonyl chloride, etc. may also be conveniently used. The nryl protecting group of compound (6) is removed in dilute hydrochloric acid or in the presence of fluorine ions to obtain H6-penzoyl-2'-deoxyaristeromycin (7).Then, the hydroxyl group at the 6' position is removed. is protected with DMT.This DMT protector (8) is treated in the presence of dimethylaminopyrimidine (DMAP) (2,3
Succinic acid anhydride is allowed to act on the hydroxyl group at position '' to obtain a succinic acid monoester (9). PY represents pyridine. Next, 2'-deoxyaristeromycin derivative 1 of general formula (II)! ! : The spacer is fully closed at the s'-terminus and bound to a support for solid phase synthesis. Here, the carrier itself is
Any conventional solid phase synthesis method for oligodeoxynucleotides may be used, for example, an acrylamide carrier (e.g., polydimethylacrylamide, polyacrylmorpholide, etc.), a folystyrene carrier (e.g., III
)-C6H4CH2NH2,. indicates a polymer), cellulose carriers (eg, aminoethyl cellulose, etc.), silica gel carriers, and the like. The compound of general formula [■] is deoxyadenosine (
: Known methods in K., Miyoshi et al., Nucl.
. Ac1dsRes., 8.5473 (1980)], the carboquine group of the spachy residue is converted into an active ester such as pentachlorophenyl or p-nitrophenyl in the presence of DCC, and then reacted with the amino group of the carrier. Next, the oligodeoxynucleotide in which part or all of the 2'-deoxyadenosine referred to in the present invention is replaced with 2'-deoxyaristeromycin is prepared by the following method (prepared from 2). For example, an oligodeoxynucleotide having a 3' terminus (22'-deoxyaristeromycin) can be obtained by converting the 2'-deoxyaristeromycin derivative of the general formula [lIJ] obtained by the above method to the 3' A conventional solid phase synthesis method for oligodeoxynucleotides (for example, Chemical and Enzymatics, Inc. Gene Fragments; A Laboratory Manual; Edited by Anne Lang
andEnzymat+c 5ynthests of
(3ene Fragments. A Laboratory Manual, FJd
tted by HoG. Ga55en and Anne Lang,
Weinheim-Deer-field Beac
h, Florida-Basel ・1 g B 2
, pp. 81-102)]. That is, when the 6'-position hydroxyl group of 2'-deoxyaristeromycin in the support for solid-phase synthesis of the present invention is protected, an acid (e.g., benzenesulfonic acid, trichloroacetic acid, dichloroacetic acid, etc.) can be prepared by a conventional method. ) to remove the protecting group and wash well with dichloromethane etc. To this 6'-position hydroxyl group, add a condensing agent (e.g., menthylenesulfonylnitrotriamine) to the target oligodeoxynucleotide base sequence (: Therefore, the mono-trimer from which the 3'-position phosphoric acid protecting group has been removed) is added to the 6'-position hydroxyl group. zolide, menthylenesulfonyl tetrazolide or menthylenesulfonyl chloride and N-
The reaction is carried out in the presence of a mixture of methylimidazole and the like. Next, the unreacted 6'-position hydroxyl group is acetylated with acetic anhydride in pyridine in the presence of dimethylaminopyridine. By repeating this operation, approximately 2-150-mer fU godeoxynucleotides having arbitrary nucleobase sequences can be synthesized. In order to cut the desired amount of sesame from the carrier, it was treated in a solution of pyridine-2-aldoxime and tetramethylguanidine at 40°C overnight, and then P was removed from the carrier. Concentrate and heat in concentrated aqueous ammonia for 60' (::, 4 hr to remove acyl protecting groups. Crude product 4MMC-GE
After rough purification on L (Yamamura Kagaku Kenkyu Kenkyutsu, 0DS1-40/46), ion exchange high performance liquid chromatography (
Gilson, Par-tisil 10 SAX)
After separating the oligomers from the top and desalting them,
Remove the DMT protecting group in 0% acetic acid. The desired 76f oligomer was then purified using reverse phase high performance liquid chromatography (Nucleosil, Cl8). Next, in order to produce the oligonucleotide by a liquid phase method, for example, N6-penzoyl-2'-deoxy-6'-0-(4,4'-dimethoquinotritino2 aristelomynon-3 '-phosphoric acid-(β-cyanoethyl, parachlorophenyl) ester or 2'-deoxyaristeromycin (2 is N
6.02'-Dibenzoyl-21-deoxyaristeromycin All conventional oligodeoxynucleotide syntheses as well as fully protected monomers (- are used and known methods (
J., Stawinski. T, Hozumi, S, 'A, Navang, C
, P., Bahl, R. Wu, Nucleic Ac1de Re5earc
h, 4, 353 (1977)). (See the margins below.) Synthesis of N6-benzinogelisteromycin (3) Aristeromycin (9.46 g, 85.7 mmol) was diluted with 100 g of dry pyridine, and after azeotropic dehydration, 150 g of Benzoyl chloride (23yil, 0.20mol) was dissolved in dry pyridine and stirred at room temperature for 2 hours.25d of water was added to the reaction mixture and stirred at room temperature for 2 hours.The reaction mixture was concentrated under reduced pressure to obtain a solid. After dissolving in CHCl3 (300 ml), washing with water, and drying (anhydrous sodium sulfate), CHCI3 was distilled off under reduced pressure.The obtained yellow solid tetrabenzoyl compound (2
) to 200g/pyridine, 150m/(7)THF
, O and 100 mlO) MeOH (: dissolved 50 d
2N NaOH was stirred at the processing room temperature for 2 hours. 85+w/])owex-50(H) to the resulting red solution
After neutralizing with water, filter the resin and dissolve the resin in water/MeOH
= 1/1 (v/v) The washing filtrate and washing liquid were combined with 100'ml and concentrated under reduced pressure. The precipitated white precipitate was collected by filtration, washed with water and acetone, and air-dried (10.82 g).
Compound (3.) was obtained as a white powder. part of gtO
Recrystallization from H gave colorless columnar crystals. ml) 199
-200'CNMR (60MHz, DMS O-d
61 Jpl) m :2, + (8H, H4", H5'
), 3.5 (3H, m, H6+. -OH), 4.2-5.1 (5H, m, H1',
H2'. HB= , -0Hx2), 7.44 (3H, m)
, 7.97 (2H, m), 8.50 (LH, S), 8.
65(11(,S), 10.0(1)1.br) Example 2 Synthesis example of N3-benzoyl-3',σ-0-(tetraisoprobyldisiloxanyl)aristeromycin (4) The benzoyl derivative (3) obtained in 1 (8.36 g, 2.6 mmol) was suspended in 80 ml of dry pyridine and dehydrated under reduced pressure (double azeotropic dehydration).Then, it was dissolved in 1001 t of dry pyridine, and 1.3-cyclo1 .1.8.3
-tetraisopropyldisiloxane (9,419,80
, 8 mmol) and stirred for 2 hours at room temperature.The reaction solution was stored in the refrigerator overnight, and the solvent was removed under reduced pressure to obtain a yellow syrup-like substance.
After partitioning between +t ethyl acetate (FJt.Ac) and Loom/water, the aqueous layer was
Extracted with gtOAc of OL+/. Combined EtOAc
The solution was washed with water (5 (++/XS), dried (anhydrous sodium sulfate), and concentrated under reduced pressure to obtain about 15 g of yellow syrup. /MeOH=50/1
．． 25/1) to give a pale yellow glassy compound (
4) (10.5 g) was obtained. A small amount of bis isomer was obtained as a by-product. NMR (60MHz, CDCl3)Jp[)m:
1.1 (28H, m), 2.23 (3H, m, H4
1, H, l). 846(1)1. br, 2°-OH1, 8,96 (2H
. m, H6*), 4.46 (tH, m, H2+
), 4.78 (2H, m, H1+, H3')
・, 7.5 (3)1, m). 8.04 (8H, 5and m, H2or HB)
. 8.67 (IH,s, H2or Hg), 9.72
(LH, br, NH) Example 3 H6-penzoyl-3',6'-0-tetraisopropyldisiloxanyl-2'-〇-((imidazo-1-yl)-thiocarbonyl 1 alisteromycin (5 ) Synthesis of silyl intermediate (4) (3,279, IJmmol) was dissolved in 80 ml of dry dichloromethane to give thiocarponyl diimidazole (3,4 g, 18.8 mmol).
1) was added and stirred at room temperature for 2 hours. The solvent was removed under reduced pressure and the resulting residue was 100ml. of EtOAc
and 50w+l of water, and the aqueous layer was diluted with 100ml of EtO.
Extracted with Ac. Combine the EtOAc solutions and wash with water (50
After drying (anhydrous sulfuric acid), the residue was concentrated under reduced pressure to obtain a yellow glassy substance. This was purified on silica gel chromatography (70 g, solvent, CHCl3 and CHC13/
The mixture was purified with MeOH=5071) to obtain pale yellow glassy compound (5) (3, tsg). NMR (60MHz, CDCl3) Jppm
: 1.0i3 (28H, m), 2.2-2.5 (8H,
m. H4・* Hs'L 3.88 (2H, m, H6・
), 4.7-5J (2H, m, Ht', Hsl), 5
．． 83 (IH+d xd, J = 2 and 6
H2, H2-), 6.85 (IH, m), 7.37 (
4H, m), 7.82 (IH. S), 7.83 (2H, m), 8.12 (IH, m)
. 8.43 (IH, S ), 9.23 (IH, br ) Example 4 Synthesis of H6-penzoyl-3',6'-0-tetraisoprobyldisiloxanyl-21-deoxyaristeromycin (6) 2 '-Thiocarbonyl body (5) (3.15g, 4.4m
mol) was dissolved in 50 ml of dry dioxane and concentrated under reduced pressure (to remove CHCI).B Next, it was dissolved in aolt of dry dioxane and heated under reflux to dissolve tributyltin hydride (4.24 mmol, 14.6 mmol) in dry dioxane (1011 mmol). The solution was added dropwise. On the way a, a'-
Crystals of azobisisobutyronitrile (AIBM) (60
09) was added little by little. The dropwise addition was completed in 30 minutes, and reflux was continued for an additional 1.5 hours. Decompression. After removing the solvent, the resulting oil was washed with 200g+/gtOAc+2-dissolved water (5(1+tX4)), dried (anhydrous sodium sulfate), and then concentrated under reduced pressure to obtain a bright red syrup-like substance.This was purified using silica gel. A colorless glassy compound (6) purified on chromatography (40 g, solvent: CHCl3)
(1.96 g) was obtained. NMR (60MHz, CDCl3) lJppm:
1゜07(28H,s), 1.8-2.5t5H,m
. H2', H4', H5'), 3.8
0 (2B, m, H6・entering 4.3 5.2 (2H,
m', H4・, H3'), 7.23 (3H, m
l, 7.77 (LH,S), 7.80 (2H.m), 9.60 (LH,br, INH) Example 5 Synthesis of H6-penzoyl-27-deoxyaristeromycin (7) a) Implementation The compound obtained in Example 4 (1.96 g of 6N, 3.3 mmol) was dissolved in 80 ml of tetrahydrofuran (THF) and mixed with 5 sl each of 1M tetraethylammonium bromide solution (5 Jl/) and IMKF solution (5 ml).
) and heated at 40-45°C for 4 days. The solvent was removed under reduced pressure and the resulting oil was converted to 50 d of water, CHC! 3
(30m1×3) After drying the organic layer (
Anhydrous sulfuric acid (IJum) was concentrated under reduced pressure to obtain a colorless syrup-like substance. This was subjected to silica gel chromatography (309, solvent, CHCl, and CHCl3/MeOH=250/
15.25, 85.50) to obtain 014 g of a colorless syrupy substance. Based on the behavior on TLC and NMR spectrum data, this compound is the target compound (7), and is considered to be 2'-deoxyaristeromycin, which was produced as a by-product during the reduction. and tributyltin hydride. NMR (60 MHz, CDCl3) appm: 2.
0 -2.5 (5H, m, H2-, H41, H
6, 1.68 (2H,m, H6・), 4.37 (
aH, br, H3'. OH), 4.8-5゜2 (LH, br, Hl・),
7.27 (3H, m), 7.80 (2B, m), 8.1
2 (IH,S), 8.45 (IH,S),
9.5(IH,,br)b) 2'-deoxyaristeromycin (500jlF) was suspended in dry pyridine 1(1+l), benzoyl chloride (2Ilt) was added, and the mixture was stirred at room temperature for ahr.The reaction solution was heated to 40°C. After concentrating to dryness, the residue was dissolved in 20 g of CHCl3 and washed with water.
．． Afterwards, it was concentrated to dryness. The solid was hydrolyzed with 2N NaOH in the same manner as in Example 1 to obtain H6-penzoyl-27-deoxyaristeromycin (7) (7) as a white powder.
Obtained 480 capes. C) Thiocarbonyl body (5) (10,839,15,2
mmol) in 100 ml of dry dioxane (2) and heated under reflux (nitributyltin hydride (14,19
+1'. A dioxane solution (48.8 mmol) and AIBN crystals (1.82 g) were added little by little over 30 minutes, and the mixture was further heated under reflux for 30 minutes. After adding dropwise 465 ml of concentrated hydrochloric acid to the reaction solution, it was left to stand at room temperature for 30 minutes. Baking soda (
After addition of 2.5 g), the solvent was concentrated under reduced pressure, and the resulting mixture of oil and aqueous solution was extracted with CHCl3. It was confirmed by TL (' that the target (7) has two aqueous layers. This aqueous solution was passed through a 2709 activated carbon column to adsorb the target substance, and after washing the column with water, acetone/water = 4
Elution at /1 (1,51) yields a small amount of 2'-deoxyaristeromycin. Then, when eluting with pyridine and water = 1/1 (1,51), the target compound (
7) was obtained. Since the obtained compound (7) was colored brown, it was decolorized on HP-20 resin. Crystal 2489 was obtained. Pale yellow columnar crystals mp 174-175°C (from EtOH) Elemental analysis value Cts) (ts N5o, , molecular weight 858.
a as 9. Calculated values: C, 61, 18, Ni 5.42. Ni19.8
2° Actual value: Ci 61.08. Hi 5.49. N,
19.71゜Example 6 H6-penzoyl-21-deoxy-6'-0-(4,
4'-dimethoxytrityl) alisteromycin (8)
Synthetic deoxy form (7) (1,25, 8.5mm01) of 2
The mixture was suspended in 0 ml of dry pyridine and azeotropically dehydrated under reduced pressure. Next, 4.4/-dimethoxytrityl chloride (1.46 g, 4.3 mmol) was dissolved in 15 g of dry pyridine and reacted for 4 hours at room temperature. IMt water was added to the reaction solution and concentrated under reduced pressure. The yellow oil obtained was dissolved in 100 g+/EtOAc, washed with water, dried, and the solvent was removed under reduced pressure to obtain 2.2 g of a yellow oil. This material was chromatographed on silica gel (409, solvent, C
HCl3 and CHCl3/MeOH=80
/1. ) to obtain 1.5 g of compound (8) as a yellow oil. NMR (60MHz, CCDCl5) 1pp: 1.
8-2.5 (5H, m,) (2+, H4-, H5
-), 3.78 (6H,S), 3. '82 (2H,
m, Hc), 4.3-5.3 (2H, m, H4
I, Ha・), 6.7-7.2 (8H, +n), 7
．． 27 (8H, s and +71), 8.15 (LH, s
), 8.48 (IH, S), 9.6 (IH, br) Example 7 H6-penzoyl-2'-deogycy 6'-〇-(4,
4'-dimethoxytrityl) alisteromycin-3'
-Synthesis of monosuccinic acid ester (9) Succinic anhydride (1
,12+1. 11.2 mmol), dimethylamino r° lysine (1.34 g, 11.0 mmol) and the deoxy compound (8) obtained in Example 6 (3.23 g, 4.g
mmol) was dissolved in 16 liters of anhydrous pyridine, and the reaction was carried out overnight at room temperature. After concentrating the reaction solution to about 1%, acetone and water were added to the extent that it became slightly cloudy, and Lich
Purified on a column of roprep RP-8 (60 g). Acetone/water = 15/85 (v/v), 2
Wash the column in the order of 5/75 (V/V) (both containing 0.1% pyridine) and then 60/40 (V/V,
The target product was eluted with 0.1% pyridine (containing 0.1% pyridine), and the solution was concentrated to dryness. The resulting oil was dissolved in a small amount of dichloromethane, the solution was added dropwise to hexane, and the resulting precipitate was collected. Vacuum drying yielded 8.209 of the 2'-deoxyaristeromycin derivative (9) of the present invention. Example 8 N-benzoyl-2'-deoxy-6'-0-(4,4
'-dimethoxytrityl) alisteromycin-3'-
Synthesis example of succinic acid pentachlorophenyl ester (10)? 2'-deoxyaristeromycin derivative (9) (, 42 (11 #g, 0.5 mmol) obtained in
and pentachlorophenol/l/(140 da, 0.5 mm
ol) was dissolved in 5Nt of dichloromethane (dichloromethane), cyclohexylpropylene (DCC, 201)q, LOm
mol) was added and the reaction was carried out overnight at room temperature. After removing the precipitated synchhexylurea P, the P solution was concentrated under reduced pressure to obtain a brown oil. After adding a small amount of toluene and removing insoluble matter, this was added dropwise to hexane, the resulting precipitate was centrifuged and dried, and the desired compound (10) was powdered 58011
(Contains 9 dicyclohexane: ¥'''). Example 9 Supported on aminomethylated polystyrene resin to preserve fc
Production of J2'-deoxyaristeromycin Aminomethylated polystyrene resin (NH2 content 1: 0.63m
mol/g, Protein Research Grant) 2.709 (1,
Active ester 580 obtained in Example 8 with 7mmo-11
"f" was mixed in 20 ml of dry DMF', triethylamine/<O, L4d, 1.0 mmol) was added, and the mixture was stirred at 25-28C for 18 h.
DMF, pyridine IIQ (wash twice, then 25j/
The free amine groups were protected by stirring in anhydrous pyridine, 5 ml of acetic anhydride, and 1 ml of triethylamine at 25-28°C.The resin was washed with pyridine, dichloromethane, and ether in this order, and then dried in vacuo. The polystyrene resin supported on the resin and containing the spacer is represented by Ar(Ar), where Ar is ? - Indicates that it is deoquine alisteromycin. Example 10 5'GTCCTGGCATGCACTTGCAR 8'
0) DMT-λr-@254 obtained in Solid Phase Synthesis Example 9 was placed in a solid phase synthesis reactor (=) and contacted with a dichloromethane solution of 3% trichloroacetic acid (TCA) to form dimethoxytrityl at the 6' position ( DMT) group was removed, and the 6' position was restored with a hydroxyl group (di-restored.) This DMT deprotected product was added with iB Bz DMTGCru φC1 (iB represents an inbutyryl group)
Cyanoethyl 3' phosphoric acid ester (from which the CBI protecting group was removed under basic conditions) was condensed by reacting at C140'C for 20 minutes in the presence of 301'M of menthicinsulfonyl-3-nitrotriazolito (MSNT). The thus obtained DMT GlBC''Ar -@ was treated with a TCA solution to remove the DMT group at the 5' position, and then the next dimer floc TT was condensed in the same manner.Hereinafter, using this method,
The dimer blocks are condensed in the order of AC, GC, AT, GC, TG, CC, and GT to form DMTG1BTcB2cB.
2TG1BG1BcB2AB2TGIBden ABZCBZ
Trrol BcBZA, BZ-◎ was obtained. After treatment with 1.8 ml of a mixed solution of 0.5 M pyridine aldoxime and tetramethylguanidine, the resin particles were removed. This oligomer solution was heated in concentrated ammonia water at 60°C for 4 hours to remove each protecting group on the base moiety. The reaction mixture was purified by ion exchange high performance liquid chromatograph 77 (-[1'artial 108AX, solvent: 0,3
M KH2PO4 solution (pH 6,3)] by linear concentration gradient method. After desalting the slowest eluting target fraction, the DMT group was removed with 80% acetic acid, and the DMT group was removed using reversed-phase high performance liquid chromatography (-[Nucleosi
l 5cts, solvent: 0.1M containing s% CHBCN)
ethylamine-acetic acid (TBAA) solution and 40% C
0.0 containing H3CN. The desired oligomer (, 1
4QJzso) was obtained. Example 11 Synthesis of Nru I linker (AATTTCGCGAr) The above oligomer was synthesized in the following order in the same manner as in Example 10 using MT-Ar-125 da, a commercially available protector and dimer. Synthesized. 5'AATTTCGCGAr 3' (a) Reversed phase and ion exchange high performance liquid chromatography (HPLC)
The product was purified using 30(IJ)2rso to obtain the desired product. (a) in solution, 5'AATTTCGCGAr a'3'
It is clear from the reference examples described below that ArGCGCTTTAA 5' becomes a partially self-complementary strand and can be inserted into the gcoRI cleavage site of the plasmid. Example 12 Phosphorylation of the 5' end of oligomer (a) 5 μl (1 μ9/μl) of an aqueous solution of C) was mixed with 6 μe of phosphorylation buffer (0.5M Tris-MCI pH 7.6, o,
tM MgC + 2.0.1M 2-mercaptoethanol) and 39 gg of distilled water, heated at 70°C for 2 minutes, then cooled to 37°C (secondary).
l of 1mM ATP, 311g(7)T4 po
lynucleotidekinase (manufactured by Takara Shuzo)
, and 1 pe of distilled water were added to give a total solution of 60 μg. After heating the reaction solution at 37°C for Ihr, 40 μl
diluted with distilled water. Example 13 N6. o Synthesis of 2'-dibenzoyl-2'-deoxyaristeromycin 1.25'i (154 mmol) of N6-penzoyl-2'-deoxyaristeromycin was dissolved in dry pyridine (15 g/) and diluted with 4,4'-dimethoxy Trityl chloride 1.469 (4.31 mmol) The whole process was stirred for 4 hours. 0.9'1 of benzoyl chloride (
6, g mmol) was added thereto, and the mixture was further stirred for 2 hours. The reaction was stopped by adding 2 ml of water, the solvent was distilled off, and 50 ml of water and 50 ml of ethyl acetate were added to the resulting oily substance and the mixture was shaken. The ethyl acetate layer was further washed twice with 50ml of water,
The ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The obtained oily substance was dissolved in chloroform (20 liters
), acetic acid (5 liters) was added thereto, and after stirring for 1 hour, chloroform (40d) was added and washed with water. The chloroform layer was chromatographed on silica gel (509, solvent; C
Purification with HCla/MeOH2O: 1) gave the desired product (1.20 g) as a light yellowish brown foam. Example 14 N6-penzoyl-2'-deoxy-6'-0-(4,
4'-dimethoxytrityl) alisteromain-3'
-Synthesis of phosphoric acid (β-cyanoethyl, parachlorphenyl) ester P-chlorophenyl phosphoric acid dichloride (4,969,2
0 mmol),? ethylamine (5,8 t. 40 mmol) and s-triacyl 2.76 g
, 4gmmol) in dioxane (40a+t), and the precipitated triethylamine hydrochloride was removed, and the liquid was
N6-penzoyl-2'-deoxy-6'-0-(4,
4'-dimethoxytrityl) alistelomy: /7 (6
, 559, 10 mmol) in pyridine (20 mmol). One hour later, additional β-cyanoethanol (6 ml) and 1-methylimidazole (1,889
, 23 mmol) and left at room temperature for 20 hours. The reaction solution was concentrated, and the resulting syrup was mixed with acetone (201I
Add I/) and make a small amount of water cloudy, jLich
roprep RP-8 (25-40 μm) [60
g, Merck & Co.] column. First, acetone
The column was washed with water (8 ni, 20(]+/), and then the fractions eluted with acetone-water (1:1.300/) were concentrated to dryness. The residue was dissolved in a small amount of chloroform. The white powder (6 g) obtained by submerging the dimethoxytrityl cation in n-hexane exhibits the orange-red color of dimethoxytrityl cation under acidic conditions. (yield 67%). Example 15 5'CGTArGCArTGC8' (7) C''@ (3oIIy, manufactured by Wako Pure Chemical Industries, Ltd.) held in synthetic polystyrene was placed in a solid phase synthesizer, and 3 % trichloroacetic acid (TCA) in trichloromethane.
3′- of dimethoxytrityl M (DMT) shown at position ′
The cyanoethyl group on phosphoric acid was removed under basic conditions, and the product was reacted at 40° C. for 20 minutes in the presence of 80 ml of mesitylenesulfonyl-3-nitrotriazole (MSNT). The thus obtained DMT TG18C"2-@ was treated with a TCA solution to remove the DMT group at the 5' position, and then a dimer block or compound was condensed in the order of 1, T, and CG to form DMT08201BT store G18C. ”
2A〒TG18CB2-■ was obtained. Add this to a mixed bath of 0.5M pyridine aldoxime and tetramethylguanine. After treatment with (1.8 g+/), the resin particles were removed. The solution containing the oligomer was concentrated to dryness and heated in concentrated aqueous ammonia at 60° C. for 4 hours to remove the protecting group of the base moiety. The reaction mixture was chromatographed on an ion-exchange high performance liquid chromatograph (-[Partisil 108AX.
MKH2PO4 solution (PH6,3)] was purified by linear concentration gradient method. After desalting the slowest eluting fraction, the DMT group was removed with SO'26 acetic acid and subjected to reverse phase high performance liquid chromatography [1'J'ucleos
5C18i solvent: 5% CH3CN, 1
M triethylamine-acetic acid (TEAA) solution containing 40% CtI3CN 0. IMT
It was purified on elution 1 using the linear concentration gradient method of i'+kA solution to obtain the desired oligomer (6 points D260). This product was tested using the Maxam-Gi 1bert method [A, Ma
xam, W, G11bert TsukeProc, Nat, A
cad, Sci, USA, 7th Sat. 560 (1977))
As a result of analysis, no co-oligomer band was detected in each lane (1ane) at the corresponding chain length position of Ar, indicating that Ar does not undergo any deoxynucleotide chain cleavage reaction in the Maxam-Gilbert method. It was confirmed. Reference Example 1 Digestion of pBR322 with EC0RI 15 μl (1.5 μg) of a commercially available solution of pBR322 was
pl buffer (IM NaCI, 0.5M T
r 1s-HC'l pH7,5,0,1MMgCI2
．． 10mM DTT), 3ttll(DEcoRI
, mixed with 10 pJ (7) of distilled water and heated at 37°C.
Warmed for hr. EcoR1 was then treated with phenol and the digested DMA was precipitated with EtOH.
pBR322 cut at the site was collected. Reference Example 2 Construction of new plasmid pBR2552 Linker solution obtained in Example 12 4 μ# (0.2 μg)
And the solution of DNA obtained in Reference Example 1! (7,5ttl. 0.6ttE) was added to 2 μg of Ba'77-(0,66M T
ris-HCI! pH7,6,66mM MgCl2
, o, tMDTT), 2ttl of 10 mM AT
P, aplIO) T4DNA Iigase,
and 1.5 plL7) Mix with distilled water and incubate overnight at 14°C to remove parent plasmid pBR255.
I got 2. Reference Example 3 pBR2552 Escherichia coli [E, coli DH1 (c
The solution containing plasmid pBR2552 obtained in Reference Example 2 was transformed into E. coli DHI (competent c
A solution containing 100 IIN (IXIOce
lls) and then treated at 0°C for 30 minutes, at 42°C for 90 seconds, and then at 0°C for 2 minutes. 800 for this liquid
μg (7) E, coli DHI culture solution 37
The cells were cultured for 1.5 hours. The culture medium 1 obtained here was placed on an agar medium (L-1 containing 0, III/d ampicillin).
broth) and kept at 37°C overnight to grow a colony. When 60 colonies were selected from a large number of grown colonies and subjected to mini-screening using the alkaline method, 38 clones were discovered that gave NruI fragments of approximately 980 bl) and approximately a4oobp. Reference Example 4 Large-scale isolation of new plasmid pBR2552 and digestion with various restriction enzymes Colonies selected based on the results of the mini-screening performed in Reference Example 3 were cultured in large quantities (L-broth
, containing 0.1 Mf/ygl of ampicillin), centrifuged and collected cells were treated with lysozyme and RNaseA.
and then ultracentrifuged in a cesium chloride solution (5500
Plasmid pBR2552 was isolated by 0 rotations for 17 hours). After extracting ethidium gromide used for color development with butanol, 10mM Tris-HCe (
Dialysis was performed in an aqueous solution le containing 1 mM EDTA (pH 8) and 1.3 ml of the plasmid solution (1
0 μl/μe) was obtained. The plasmid thus obtained was digested using a combination of several restriction enzymes, and the behavior of the cut fragments was observed during electrophoresis in agarose and polyacrylamide gel soil.
(see table), the desired prosmid pBR2552 (first
It was confirmed that the following results were obtained. Table 1: Digestion of pBR255'2 with various restriction enzymes and H for comparing pBR322 and I) BR2552.
Digestion experiments with ael and Hae[1-Nrul were performed. In pBR322, the length of the Hae[l fragment containing the EcoRl recognition site is 192 bp. Since pBR2552 cut out the EcoRl recognition site and newly incorporated a linker for Nrul (10mer), a 192 bp fragment was not obtained, but a 202 bp fragment should have been obtained. This was confirmed by the behavior of the digested product on polyacrylamide gel electrophoresis (Figure 2). In addition, the above 202 bp and 51 bp fragments (pBR255
2) has a recognition site for Nru l, so it is Ha
After digestion with e l -Nru l, it becomes even shorter fragments (2o2bp - 180bp + 22bp, 51bp
-8tbp+2obp). ,? J) Cotomo polyacrylamide gel electrophoresis behavior confirmed (Fig. 3) Accordingly pB R322 (7) Linker for Nrul containing Ar in the EcoR1 digestion site (A, 10mer)
It is clear that only - pieces of . Effects of the Invention The 27-deoxyaristeromycin derivative of formula 14 (11) of the present invention is a chemical compound of oligodeoxynucleotides having 2'-deoxyaristeromycin instead of 2'-deoxyadenone as a constituent nucleoside. In other words, in the chemical synthesis of oligodeoxynucleotides having a 2'-deoxyadenone, depurination of the nucleoside occurs under acidic conditions such as deprotection, but the 2'- This disadvantage is not observed when using deoxyaristeromycin.
When producing an oligomer in which 2'-deoxyaristeromycin is located at the 'terminus' by solid-phase synthesis, the 2'
Although deoxypristeromycin is exposed to acidic conditions very frequently, depurination is not observed, and solid-phase synthesis can be advantageously applied. The oligodeoxynucleotide thus obtained can be used in genetic engineering in the same manner as the oligomer that injects deoxyadenone, as shown in the reference example above. In other words, the oligodeoxynucleotide of the present invention can be used in a vector into which the oligodeoxynucleotide of the present invention has been inserted. is recognized by microorganisms in exactly the same way as those containing deoxoadenosine, and during plasmid replication (= is replaced with deoxyadenonine, which is very convenient.

[Brief explanation of the drawing]

Figure 1 shows the restriction enzyme map of plasmid pBR2552, and the numbers in the figure represent EC0RI in pBI (3221=
Shows the number of bases from. Figure 2 shows pBR322 and p
BR2552 was digested by Hae, and also the third
The figure shows the electrophoretic behavior of the digested product on a polyacrylamide gel [i] when digested with Hae[[-Nrul].

Claims (1)

[Claims] 1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R^1 is hydrogen or an amino group protecting group, R^2
is hydrogen, a spacer residue, a hydroxyl protecting group, an optionally protected phosphoryl group, or an optionally protected deoxynucleotide chain bonded at the 5' position, and R^3 is hydrogen, a hydroxyl protecting group, or an optionally protected deoxynucleotide chain bonded at the 3' position. indicates an optionally protected deoxynucleotide chain bonded with R^1, R^2
and R^3 are hydrogen at the same time) 2) General formula ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ (In the formula, R^1 is hydrogen or amino The group protecting group is R^2
is hydrogen or an optionally protected deoxynucleotide chain bonded at the 5' position, and R^3 represents hydrogen, a hydroxyl group-protecting group, or an optionally protected deoxynucleotide chain bonded at the 3' position. 1. A carrier for solid phase synthesis of oligodeoxynucleotides, comprising a 2'-deoxyaristeromycin derivative bound at the 3' end via a spacer. 3) Part or all of 2'-deoxyadenosine is converted into 2'
-Oligodeoxynucleotides replaced with deoxyaristeromycin.